Mold culture



March 31, 1942.

Filed June 16, 1939 mom ATTORNEY.

' values.

Patented Mar. 31, 1942 MOLD CULTURE Gilbert B. Ayres, Stamford, and Joseph George Niedercorn, Riverside, Conn., American Cyanamid Company,

assignors to New York,

N. Y., a corporation of Maine Application June 16, 1939, Serial No. 279,470

l Claim. The invention relates to the controlled digestion of protein materials and more particularly to a the bating of hides and skins by subjecting them to the action of tryptic enzymes of Aspe'rgz'llus'.

fumigatus.

It has been discovered that Aspergillus fumiuniformly good rate of activity in alkalies at least up to the point where the alkalinity reaches a pH value of about 9.5.

The preparation of these enzymes is as follows: A suitable nutrient medium in the granular or solid discrete particle condition, such as gatus produces enzymes which have sufiicient proteolytic activity under neutral, acid and alkaline conditions to render them commercially attractive in the bating of hides and in similar processes involving the selective digestion of proteins.

It is an object of the present invention to provide these enzymes in the form of a dried pro-. teolytic culture which is suitable for use as a hate. It is a further object to provide a method for the bating of hides and skins using these enzymes as bates, preferably in conjunction with a deliming agent such as ammonium sulfate.

The invention includes both the production of these enzymes from Aspergz'llus fumigatus by inoculation of a suitable nutrient medium and cultivation of the inoculated medium and the provision of the enzymes and nutrient medium or carrier in dried form, and also the selective digestion of proteins and the hating of hides and skins by subjecting the materials to the action of these proteolytic enzymes.

As illustrative of the proteolytic activity in alkaline solution of these enzymes produced by Aspergillus fumigatus there is shown in the single figure of the accompanying drawing a curve, the ordinates of which are given in terms of standard alkali and the abscissae in terms of pH The curve was constructed from values obtained in digesting a casein substrate (sodium caseinate) with a culture of these enzymes at increasing alkalinities of the sodium caseinate solution. The undigested casein was precipitated with a standardized solution of sulfuric acid and sodium sulfate and the filtrate therefrom, containing the digested casein, titrated with 0.1 N-alkali. The tests were conducted by a modification of the procedure of Volhard and Loehlein for the determination of proteolytic activity of enzymes described in Praktikum der Physiologischen Chemie," part 1, pages 258-259, by Peter Rona, 2d Ed., Julius Springer, Berlin, 1931. The curve which was plotted for the pH range of 7-10,

after sloping oil from the point of neutrality,

changes sign and ascends until a pH of about 9.5 is reached whereuponit again changes sign and slopes downwardly to the pH of 10. By inspection it will be seen from this curve that the enzymes of the present invention manifest a bran, moistened withan equal weight of water, is inoculated with a culture of Aspergillus fumigatus and the inoculated moist bran spread out in thin layers on trays. The inoculated bran is then incubated in an oven maintained at a temperature of about 30 C. and preferably at not higher than this temperature, and at a humidity in the oven such that the atmosphere therein is saturated but does not contain suflicient moisture to cause deposition of the same onto the bran. The inoculated bran is maintained in the oven until sporulation occurs. After incubation for the optimum period the bran culture may be thoroughly mixed with 0.2% of cresylic acid in solution, if desired, to improve the wetting out of the bran when used in solution. The culture is then dried ata temperature below 45 0., es. 40 C., and may be used as such for hating, or an ammonium salt, e. g. ammonium sulfate, may be incorporated with the moist mass and the mixture then dried.'.--The bran used for the culture may or may not be sterilized before the inoculation and likewise the culture may or may not be sterilized, although sterilization of the culture does not appear necessary at the present time.

The enzyme may be liberated from the dried culture by elution withdilute solutions of various salts, such as ammonium chloride, ammonium sulfate, sodium chloride, sodium sulfate, etc. and the eluted enzymes used in other fields as digestants.

The proteolytic enzymes may be applied to the hating of hides and skins in the form of the combined enzymes and nutrient medium or carrier in any manner now practiced in the art for the application of other tryptic bates. One method now in use involves washing the hides from the dehairiiig step, and adding an ammonium salt, such as ammonium sulfate, to the water containing the hides in order to lower their pH which is generally very high due to the strongly alkaline conditions under which dehairing takes place. Before adding the ammonium salt the hides may be treated for re moving lime blast by the addition of a suitable acid, such as hydrochloric acid, to the water bath containing thehides. After the pH of the bath has been suitably adjusted, the enzyme bate is added thereto in anamount determined by the enzyme unit strength 01' the bate, the kind or hide or skin to be bated, the extent of bating desired, the length of the bating time and the temperature of the hating bath.

For the purpose of illustration, there is described in the following example a method for bating oi hides with the enzymes of Aspergillus fumigatus.

Example Wash a 500 lb. pack of limed kips from the dehairing bath with water for 15 minutes at 70 1''. Heat the washed pack in water to 95 F. and add 5 lbs. of hydrochloric acid thereto, the bath showing red to methyl orange. After three minutes add lime to the bath until it is slightly, pink to phenolphthalein. To the bath then add 3V lbs. 01' ammonium sulfate and about 1% (based on the weight of the kips) of dried culture prebe used in many other processes in which a tryptic enzyme of high activity is desired,.such as in desizing and degumming textiles, paper sizing, tenderizing of meat, stripping oi! gelatin from photographic films and plates, manufacture oi peptones, chewing gum, glue, foods, drugs and biological products c r in any field where by their use a protein or a protein degradation product can be reduced to a lower molecular size of increased solubility.

It will be understood that the above description is intended as illustrative and not as limiting of the invention, the scope of which is defined by the following claim.

What we-claim is:

A dried culture of uniformly high proteolytic activity in the pH range of 'l to about 9.5, said culture consisting essentially of enzymes of Aspergillus jumigatus on a bran carrier and being the product of inocculating bran as the nutrient medium with the mold Aspergillus fumigatus, incubating the culture under conditions of heat and moisture whereby the bran is impregnated with enzymes of'proteolytic activity, and drying the incubated culture.

GILBERT B. AYRES. JOSEPH G. NIEDERCORN. 

